1. Field of the Invention
The instant invention pertains to cholesterol assay enzymatic reagents containing cholesterol oxidase.
2. Description of the Prior Art
Present enzymatic reagents using cholesterol oxidase to measure cholesterol in a specimen measure the amount of hydrogen peroxide produced by the reaction between oxygen and cholesterol which is translated into a measure of the amount of cholesterol in the specimen. The hydrogen peroxide measurements are generally based upon the reaction between hydrogen peroxide and a dye which causes a change in color. This change of color is measured and is translated into a measurement indicating the quantity of hydrogen peroxide present.
Reducing substances, such as glutathione, ascorbic acid and uric acid which are present in human fluids, react with hydrogen peroxide and prevent all of the hydrogen peroxide from reacting with the dye. This results in a loss of accuracy in the cholesterol assay.
One solution to this problem is to measure the rate of consumption of oxygen in enzymatic conversion of cholesterol to cholest-4-en-3-one and hydrogen peroxide and to translate the measurement into the amount of cholesterol present. An oxygen rate analyzer and method providing such a measure are described in U.S. Pat. Nos. 3,857,771 and 3,933,593 to James C. Sternberg, said patents being incorporated herein in toto by reference.
While oxygen rate measuring systems, such as described in the aforesaid Sternberg patents, may be usefully employed to measure total cholesterol in a specimen, the presence of substances in the specimen which inhibit the reaction of enzymes with cholesterol and/or cholesterol esters or which inhibit the availability of cholesterol for reaction with the enzymes (hereinafter referred to collectively as inhibiting agents) may render such measurements rather inaccurate. In particular, inhibiting agents which appear to be present in turbid serum containing above normal concentration of triglycerides appear to decrease the availability of reactable cholesterol and/or decrease the rate at which the enzymes catalyze reactions leading to the consumption of oxygen (hereinafter referred to collectively as the oxygen consumption inhibiting effects). In either event, the result is a reduction from the normal rate of consumption of oxygen such that the measurement of the rate of oxygen consumption in the presence of such inhibiting agents does not yield an accurate measure of cholesterol concentration. For example, it has been found that when using the oxygen rate measuring system to determine the total cholesterol level in certain specimens, the rate of oxygen consumption was lower than would be expected from the cholesterol content of the specimen, the actual cholesterol content of the specimen having been previously determined by the well accepted assay for total cholesterol of Abell et al., J. Biol. Chem., 195:367 (1952), said article being incorporated herein in toto by reference.